Download Water Transport by Aquaporins in the Extant Plant

Water Transport by Aquaporins in the Extant
Plant Physcomitrella patens1[W]
David Liénard, Gaëlle Durambur, Marie-Christine Kiefer-Meyer, Fabien Nogué,
Laurence Menu-Bouaouiche, Florence Charlot, Véronique Gomord, and Jean-Paul Lassalles*
Université de Rouen, CNRS UMR 6037, Institut Fédératif de Recherches Multidisciplinaires sur les
Peptides, Faculté des Sciences Bât. Ext. Biologie, 76821 Mont-Saint-Aignan cedex, France (D.L., G.D.,
M.-C.K.-M., L.M.-B., V.G., J.-P.L.); and Station de Génétique et Amélioration des plantes,
Institut National de la Recherche Agronomique, 78026 Versailles, France (F.N., F.C.)
Although aquaporins (AQPs) have been shown to increase membrane water permeability in many cell types, the physiological
role of this increase was not always obvious. In this report, we provide evidence that in the leafy stage of development
(gametophore) of the moss Physcomitrella patens, AQPs help to replenish more rapidly the cell water that is lost by transpiration,
at least if some water is in the direct vicinity of the moss plant. Three AQP genes were cloned in P. patens: PIP2;1, PIP2;2, and
PIP2;3. The water permeability of the membrane was measured in protoplasts from leaves and protonema. A significant decrease was measured in protoplasts from leaves and protonema of PIP2;1 or PIP2;2 knockouts but not the PIP2;3 knockout. No
phenotype was observed when knockout plants were grown in closed petri dishes with ample water supply. Gametophores
isolated from the wild type and the pip2;3 mutant were not sensitive to moderate water stress, but pip2;1 or pip2;2 gametophores
expressed a water stress phenotype. The knockout mutant leaves were more bent and twisted, apparently suffering from an
important loss of cellular water. We propose a model to explain how the AQPs PIP2;1 and PIP2;2 delay leaf dessication in a
drying atmosphere. We suggest that in ancestral land plants, some 400 million years ago, APQs were already used to facilitate
the absorption of water.
Many aquaporins (AQPs) have been detected in animal cells (Agre et al., 2001; Zardoya and Villalba, 2001;
Agre and Kozono, 2003) and plant cells (Chrispeels
et al., 2001; Maurel et al., 2002; Luu and Maurel, 2005;
Hachez et al., 2006). Although several studies reported
that AQPs may increase membrane permeability to glycerol (Stroud et al., 2003), urea (Echevarria et al., 1994),
and perhaps CO2 (Uehlein et al., 2003) or even ions
(Yasui et al., 1999; Hazama et al., 2002), it is now well
established that most of these transmembrane proteins increase the water permeability of cellular membranes. Thus, AQPs are potentially playing a role in
many physiological processes (Maurel et al., 2002;
Tyerman et al., 2002; Luu and Maurel, 2005) involving
water flow through membranes. However, even in wellknown processes such as transpiration, when plants are
losing water due to environmental conditions, establishing a close correlation between AQPs and water
transport appears complicated.
During the evolution of land plants two adaptation
strategies emerged to cope with irregular water sup1
This work was supported by a grant from the Conseil Régional
de Haute Normandie (to D.L.).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jean-Paul Lassalles ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.111351
ply. A homoiohydric plant-like Arabidopsis (Arabidopsis thaliana), maintains its water status by controlling
water potential, through turgor and osmotic pressure
regulation. In this type of plant there are two parallel
pathways for water transport known as the apoplastic and symplastic routes. The relative contribution of
each pathway can vary, making it difficult to determine, with great accuracy, the magnitude of transmembrane water flow (Tyerman et al., 1999). Under
most physiological conditions, water appeared to simply diffuse across the lipid bilayer (Finkelstein, 1987)
and it has been difficult to show that water movement
requires an increase in the water permeability (Pos) of
the membrane bilayer by the action of AQPs. Hill et al.
(2004) even suggested that the main function of AQPs
is to act as sensors for osmotic and turgor pressure,
and that their capacity to conduct water is consistent
with the structure of a sensor.
We expected to find a closer connection between
AQPs and transmembrane water flow in the moss
Physcomitrella patens for several reasons. First, poikilohydric organisms such as mosses, whose water content
varies with the external environment, do not regulate
their water potential, and the need for turgor or osmotic
sensors may not be as critical. Elucidation of the function of AQPs in bryophytes may hint at their function
during the early stages of land plant evolution.
A second reason is that P. patens exhibits a simple,
nonvascular structure in two different stages of its development. This moss, which lives in the open habitats
of temperate regions, is subject to important variations
Plant Physiology, March 2008, Vol. 146, pp. 1207–1218, www.plantphysiol.org Ó 2008 American Society of Plant Biologists
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Liénard et al.
in environmental parameters (light, humidity, and
temperature). It is not a desiccation-tolerant plant and
water exchanges depend on the developmental stage. In
liquid medium, a single cell gives rise to filamentous
protonema, i.e. branched files of single cells (Schaefer
and Zryd, 2001). The protonema can only develop when
fully surrounded by liquid water. Later, cell division
continues to form the gametophore leaves, which develop in the air and form the aerial portion of the plant.
The leaves have no cuticle or stomata and are one-cell
thick. In the juvenile stage these do not possess vascular
tissue but by the adult stage a single midrib develops
(Sakakibara et al., 2003). Gametophore leaves gain or
loose water depending on the relative humidity (RH)
in the air measured far from the leaves (Nobel, 1999).
Water is mainly drawn in by the leaves from external
reservoirs through capillary action (Proctor and Tuba,
2002). It is lost through the aerial parts of the plant when
RH decreases in the air near the cell wall. During this
stage of development, water transpired from a gametophore must cross cellular membranes, so we hypothesized that AQPs could be involved in this process.
It has been known for some time that AQPs are
present in P. patens, a well-established model system
for various biological and evolutionary processes in
higher plants (Schaefer, 2002; Quatrano et al., 2007;
Shigyo et al., 2007). Twelve putative AQP sequences
were first identified in a homology screen of a P. patens
EST library (Borstlap, 2002), five of which belong to
the PIP (plasma membrane intrinsic proteins) family.
Since then, the first annotated P. patens ssp. patens genome sequence was released in 2007 (http://genome.
jgi-psf.org). Twenty-five sequences are classified into a
‘‘carbohydrate transport and metabolism’’ family with
the special annotation ‘‘aquaporin’’. Nine AQPs (including the three genes described in this article) are
considered as PIP, six others as TIP, another group of
six as NIP, two as SIP, and two are without a specific
subfamily name. A glycerol channel (Gustavsson et al.,
2005) should probably be added to this list.
In this article, we report the cloning of three putative
PIP in P. patens: PIP2;1, PIP2;2, and PIP2;3. Knocking
out (Schaefer and Zryd, 1997) PIP2;3 did not lead to
modifications in the Pos of protoplasts. On the contrary,
the PIP2;1 or PIP2;2 knockouts resulted in a significant
decrease in the Pos of protoplasts from mutant gametophores. Based on these results we conclude that both
the PIP2;1 and PIP2;2 genes encode functional AQPs.
Figure 1. The putative P. patens PIP2 genes PIP2;1,
PIP2;2, and PIP2;3. Schematic diagrams of the
PIP2;1, PIP2;2, and PIP2;3 gene structures. The numbers on the genes correspond to the first and last
bases of exons (dark boxes). The light boxes indicate
introns. Vertical bars and boxes indicate the position
of the splicing sites and transmembrane domains
within the proteins.
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Aquaporins Play Physiological Role in Physcomitrella patens
Suppression of PIP2;1 and PIP2;2 reduced the tolerance of gametophores to water stress.
RESULTS
PIPs are more similar to PIP2 than to PIP1 AQPs (data
not shown). Further molecular phylogenetic analysis
(Fig. 3) confirmed the evolutionary relationship between
these proteins. We named the three P. patens homologs:
PIP2;1, PIP2;2, and PIP2;3.
Cloning of Three AQP Genes
In the absence of the full P. patens genome sequence
(this only became available at the beginning of 2007 at
http://genome.jgi-psf.org/Physcomitrella), we searched
EST databases for sequences similar to Arabidopsis
AQPs (Nishiyama et al., 2003). This led to the isolation
of three moss orthologs from the PIP2 family. Their
gene structure was determined by comparing their coding and genomic sequences to characterize the number,
position, and sequence of the introns. The three cloned
homologs contain three introns, which is the case for all
PIP genes (Johanson et al., 2001). The position of these
introns is similar for the three genes (Fig. 1) as well as to
the Arabidopsis PIP2 genes (Johanson et al., 2001).
Open reading frames predict protein sequences of
280 or 281 amino acids. As expected for an AQP protein, six transmembrane domains (Stahlberg et al., 2001)
are visible on the hydrophobicity profile of all three
sequences (data not shown). The two NPA signature
sequences usually detected in loops 2 and 5 of AQPs
(Johanson et al., 2001) were also present in these homologs (Fig. 2). The alignment of the P. patens and Arabidopsis PIP protein sequences indicated that P. patens
PIP2;1, PIP2;2, and PIP2;3 Expression Patterns
The expression patterns of the three genes were
analyzed for two different stages of development (Fig.
4A). Total RNA was isolated from protonemata or gametophores, then reverse-transcribed. Control experiments were performed using constitutively expressed
18S RNA (Fig. 4B). We found that the transcripts were
specifically accumulated in the gametophores (Fig. 4A,
lanes g), but not detectable in protonema grown in
liquid medium (Fig. 4A, lanes p). These results clearly
showed that the transcripts are only expressed in the
aerial parts of the plant.
Targeted Knockout of PIP2;1, PIP2;2, and PIP2;3
P. patens is especially amenable to gene knockout
because of its high rate of homologous recombination
(Schaefer and Zryd, 1997). Disruption constructs for the
three genes were created by exchanging small fragments
of the genes with an nptII selection cassette (Fig. 5).
Knockout plants were screened by PCR amplification of
the nptII cassette, and the 5#- and 3#-ends of the genes.
Figure 2. Alignment of the three encoded proteins PIP2;1, PIP2;2, and PIP2;3. This alignment was made using the programs
Mutalin and ESPript (http://nosa-pbil.ibcp.fr). Predicted positions of the transmembrane domains (a1–a6) and external loops (A–E),
from N terminus to C terminus, were identical for the three sequences and are shown by the sketches on top of the sequences.
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Liénard et al.
Figure 3. Molecular phylogenic relationship
between Arabidopsis and P. patens PIPs.
The evolutionary tree was generated with
PROTDIST and NEIGHBOR of the PHYLIP
package (http://bioweb.pasteur.fr/). The bar
indicates a mean distance of 0.1 changes per
amino acid residue. Numbers correspond to
the protein identification from putative PpPIP
(http://genome.jgi-psf.org). AtPIP, Arabidopsis
PIP; PpPIP, P. patens PIP.
The knockout of each of the three genes was confirmed
by examining expression of the cDNA (Fig. 5D) in the
three mutants, pip2;1, pip2;2, and pip2;3, using reverse
transcription (RT)-PCR. As expected, the PIP2;1, PIP2;2,
and PIP2;3 transcripts were detected in the wild type
(Fig. 5D, lane WT) but not in the corresponding mutant
(Fig. 5D). Despite similarities in the PIP2;1, PIP2;2, and
PIP2;3, cDNA sequences, PCR demonstrated that each
of three gene disruptions was specific.
Measurements of Pos on Isolated Protoplasts Suggest
That PIP2;1 and PIP2;2 Function as AQPs
We measured the Pos, of protoplasts isolated from
protonema and gametophore cells (Fig. 6), as previously described (Ramahaleo et al., 1999). The data are
presented on a logarithmic scale because of the wide
range of Pos values obtained. The protoplasts from
wild-type protonema had a median value of Pos 5 2.7
mm s21 (Fig. 6A). This small value indicates simple
diffusion across the lipid bilayer (Finkelstein, 1987). In
contrast, the protoplasts from the wild-type gametophores had a median value of Pos 5 159 mm s21 (Fig.
6B). The large difference in Pos suggests that AQPs are
present in the cellular membranes of the gametophore
cells. These results are consistent with the expression
pattern of our putative AQPs described in Figure 4.
Direct comparisons of wild-type and mutant gametophore protoplast Pos values were carried out (Fig. 6).
The data did not pass the test for normality so the four
samples were compared with a nonparametric ANOVA
on ranks test followed by pairwise multiple comparison procedures (Dunn’s method). There was no statistically significant difference (P , 0.05) in the median
values between the wild type (159 mm s21) and pip2;3
(134 mm s21) or between pip2;1 (41 mm s21) and pip2;2
(32 mm s21), but the difference between wild type or
pip2;3 versus pip2;1 or pip2;2 was significant.
The decrease in Pos of more than 100 mm s21 for
pip2;1 or pip2;2, compared with wild type, suggests
that AQP function is disrupted in these mutants,
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Aquaporins Play Physiological Role in Physcomitrella patens
Figure 4. Expression pattern of PIP2;1, PIP2;2, and PIP2;3 transcripts in
P. patens wild type. For each gene, amplification with 20-mer specific
primers on genomic DNA was included as a control (A, lane G). RT-PCR
analyses were performed on RNA from 4-d-old protonema (A, lane p) or
3- to 4-week-old gametophores (A, lane g). The integrity of the total RNA
in the two preparations was assumed by checking the presence of constitutively expressed 18S RNA (B, lanes p and g). All experiments were
repeated three times with independent plant samples.
whereas the knockout of PIP2;3 has no effect on the
permeability of isolated gametophores.
Effect of Water Stress on the Gametophores
All plants, including the wild type and the three
mutants, exhibited variations in leaf size and number
when gametophores were grown in closed petri dishes.
No obvious phenotype could be identified, and withingroup variation in morphology was as great as betweengroup variation. In a set of experiments (data not
shown), petri dishes containing wild-type, pip2;1,
pip2;2, and pip2;3 plants were opened at the same
time and allowed to evaporate in a dry atmosphere
(RH, 20%–30%; temperature, 20°C–22°C). The pip2;1
and pip2;2 mutant leaves appeared to become twisted
more rapidly and in greater number than those of the
wild type and pip2;3 plants.
The effect of water stress was also studied more
directly on isolated gametophores. Three similar-sized
gametophores from wild-type, pip2;1, and pip2;2
knockout plants, were deposited on a filter soaked
with water (Fig. 7, A and B) or on a dry filter paper disc
(Fig. 7C). The moisture was allowed to evaporate in
the laboratory conditions. Gametophore leaves began
slowly moving (bending and twisting) usually several
minutes after the beginning of the experiment. These
movements are assumed to indicate significant water
loss from cells, i.e. sensitivity of the gametophore to
the stress conditions. The times, t1 and t2, recorded at
the start and finish of the movements were used as
markers for water stress. The values of t1 and t2 are
detailed in the legend to Figure 7.
When the gametophores were deposited on a dry
filter, creating what we called ‘‘strong water stress’’
conditions, the filter sucked the liquid water still present on the leaves in a few seconds and the RH near the
gametophores was probably rather low, close to that
of the environment (,60%). The stress-induced movements started and ended at the same time in both the
wild type and mutants in this experiment that is
described in Figure 7C as well as in three other similar
experiments.
We expected to create moderate water stress conditions by using a wet filter, assuming that the evaporation of water would increase humidity near the leaves.
We found that when the gametophores were deposited
on a wet filter, the wild-type or pip2;3 (data not shown)
plants remained unaffected, whereas pip2;1 or pip2;2
knockout plants moved following the same time course
in the experiment described in Figure 7, A and B, and
in three other similar experiments.
Supplemental Videos S1 to S3 (see ‘‘Supplemental
Data’’) show wilting on dry and wet filters in a typical
experiment. The strong water stress led all the gametophores to wilt at the same rate. The suppression of
AQPs increased sensitivity to moderate stress conditions in two out of the three mutants.
DISCUSSION
PIP2;1, PIP2;2, and PIP2;3 Are Expressed in Wild-Type
Gametophores and Were Specifically Knocked Out in
Mutant Plants
We screened a whole plant P. patens EST library and
identified three putative genes containing the two
NPA repeated regions considered to be characteristic
signatures of plant AQPs (Xie et al., 2003). The three
sequences also show strong similarities with AQPs of
the Arabidopsis PIP2 family (Fig. 3). We called them
PIP2;1, PIP2;2, and PIP2;3 (Fig. 1).
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Liénard et al.
Figure 5. Schematic representation of the PIP2;1, PIP2;2, and PIP2;3 genes, corresponding knockout constructs, and molecular
analysis of the knockout mutants. A, Gene structure and knockout construct of the PIP2;1 gene. B, Gene structure and knockout
construct of the PIP2;2 gene. C, Gene structure and knockout construct of the PIP2;3 gene. Dotted lines and numbers of base pair
indicate the regions of the genes used for insertion in pNeo. The position of the primers (little arrows) is detailed in ‘‘Materials and
Methods’’. D, Expression analysis of the wild-type and mutant plants. Total RNA from wild-type, pip2;1, pip2;2, and pip2;3
plants was reverse transcribed and used for PCR. The experiments were repeated three times with independent wild-type and
mutant plant samples. The integrity of the total RNAs in the preparations was assumed by checking the presence of constitutively
expressed 18S RNA.
Their expression pattern in wild type was examined
using RT-PCR and is presented in Figure 4. The PIP2;1,
PIP2;2, and PIP2;3 transcripts accumulated in the
gametophores but could not be detected in the protonema stage. This expression pattern is consistent with
a potential role as AQPs involved in transpiration.
Under normal growth conditions the protonema does
not experience water stress; it develops in dilute
solutions with a water potential close to zero. We
generated knockout mutants for PIP2;1, PIP2;2, and
PIP2;3 (Fig. 5). The control experiments (Fig. 5D)
indicated that, despite the close sequence similarity
of the three proteins, the suppression of each gene was
selective: pip2;1, pip2;2, and pip2;3 did not express their
corresponding mRNA but expression levels of PIP2;1,
PIP2;2, and PIP2;3 were normal in each of the two
other mutants.
PIP2;1 and PIP2;2 Are Functional AQPs in
Gametophore Cells
Several conclusions can be drawn from the Pos values
measured on the various protoplasts populations (Fig.
6). Wild-type protonema protoplasts had low Pos
values, suggesting an absence of active AQPs, whereas
high Pos values for wild-type gametophore protoplasts
indicate that active AQPs are functioning to transport
water across the membrane in these cells. The decrease
in Pos observed for the two knockout mutants (Fig. 6)
strongly suggests that both PIP2;1 and PIP2;2 are
functional AQPs.
PIP2;3 Has No Effect on Pos
Pos values were similar for wild-type and pip2;3
knockout gametophores. This suggests that, despite a
close similarity between PIP2;1, PIP2;2, and PIP2;3
sequences, PIP2;3 does not code for an AQP with a
significant role in water transport, at least in our
growth culture conditions (RH 5 100%). Thus, although like PIP2;1 and PIP2;2, PIP2;3 was only expressed in the gametophore, we have no indication
concerning PIP2;3 function. We could, however, consider pip2;3 as a control, because it indicates that the
recombination techniques used to obtain the mutants
are not responsible for the decrease in Pos found with
pip2;1 and pip2;2 plants.
pip2;1 and pip2;2 Plants Express a Phenotype under
Moderate Water Stress Conditions
No phenotype was observed when the knockout
mutants were grown in closed petri dishes with ample
water supply. When dishes containing either wild type
or mutants were opened simultaneously we noticed
that pip2;1 and pip2;2 leaves dried more rapidly than
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Aquaporins Play Physiological Role in Physcomitrella patens
wild-type or pip2;3 knockout leaves. Variations in transpiration rate due to fluctuations in environmental
parameters (temperature, light, and RH) are assumed
to be the same for all the plants so this did not appear
to explain the difference.
Nevertheless, to rule out the effect of local conditions, we then compared wilting of similar-sized isolated gametophores, which were deposited on the
same dry filter paper disc. Because the entire gametophores were in contact with dry air, severe water stress
was expected. In each of our experiments all the
gametophores commenced and stopped wilting at
the same time. We conclude that the AQPs do not
modify sensitivity to dehydration of the gametophores
in these conditions.
The situation was quite different when the gametophores were in contact with wet soil (water-soaked
filter paper) through some of their leaves. Less stress
was induced by these experimental conditions because only part of the leaves was transpiring in the air,
whereas the other part could absorb liquid water. In
contrast to the previous result, the water stress phenotype observed for the pip2;1 and pip2;2 knockout
mutants supports an important role for the corresponding AQPs during transpiration.
Resistance to Transpiration by a Cell Surface in the Air:
The Boundary Layer and Cell Membranes
Hydrodynamic theory describes diffusion and convection currents in a gas phase. It shows that transverse resistance to the transfer of water molecules
between a phase of liquid water and the ambient air is
mainly located within a small layer of still air near the
liquid surface, the so-called ‘‘boundary layer’’. Tazawa
and Okazaki (1997) suggested a dominant role for this
layer during evaporation from a cell surface. Based on
their experiments with Chara corallina cells, the authors
concluded that, even without AQPs, the cell membranes were much more permeable than the boundary
layer; the permeability of the lipid bilayer alone was at
least 50 times higher than that of the boundary layer.
Tazawa and Okazaki (1997) thus suggested that AQPs
Figure 6. Histograms showing the Pos values for protoplasts from
protonema and gametophores. The Pos values measured for 31 protoplasts from wild-type protonema (A) came from three different preparations. They had a diameter of: 30 6 2.5 mm. The 36 protoplasts from
wild-type gametophores (B) were from four different preparations. They
had a diameter of 49.6 6 7.8 mm. The 40 protoplasts from the pip2;1
gametophores (C) were from six different preparations. They had a
diameter of 41.8 6 4.4 mm. The 39 protoplasts from the pip2;2
gametophores (D) were from four different preparations. They had a
diameter of 47.5 6 8.5 mm. The 28 protoplasts from the pip2;3
gametophores (E) were from three different preparations. They had a
diameter of 49.8 6 5.6 mm. No correlation was found between Pos
values and the diameter of the protoplasts from gametophore or
protonema preparations. The diameter figures are given as mean 6
SD; n is the number of protoplasts in each class.
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Figure 7. Effect of water stress on isolated gametophores. The top section of the plant gametophores was deposited at time t0 on dry
or wet filter paper and exposed to a nonwater-saturated atmosphere. Times t1 and t2 correspond to initial and final leaf movements
(bending or twisting) in gametophores. Photos are shown for: t0; t2; and tm 5 (t1 1 t2)/2. A, Gametophores from wild-type, pip2;1,
and pip2;2 plants on a wet filter. The variations in environmental parameters during the experiment were: RH, 60%; temperature,
24°C. For both mutants, t1 5 45 6 5 min; t2 5 180 6 5 min. No movement by the wild type was detected. B, Gametophores from
wild-type, pip2;1, and pip2;2 plants on a wet filter. The variations in environmental parameters during the experiment were: RH,
55.5%; temperature, 28°C. For both mutants, t1 5 18 6 3 min; t2 5 84 6 3 min. No movement by the wild type was detected. In
three repeat experiments similar to those shown in A or B, the values were, respectively: t1 5 42 6 3 min and t2 5 120 6 3 min
(RH 5 60%; 28°C); t1 5 21 6 3 min and t2 5 100 6 3 min (RH 5 60%; 28°C); t1 5 36 6 3 min and t2 5 105 6 3 min (RH 5 54%;
24°C). C, Gametophores from wild-type, pip2;1, and pip2;2 plants on a dry filter. The variations in environmental parameters during
the experiment were: RH, 49%; temperature, 28°C. For both mutants and wild type, t1 5 5 6 1 min and t2 5 28 6 1 min. In three
similar experiments, the values were, respectively: t1 5 6 6 1 min and t2 5 36 6 1 min (RH 5 49%; 28°C); t1 5 20 6 1 min and t2 5
52 6 1 min (RH 5 53.5%; 24°C); t1 5 15 6 1 min and t2 5 46 6 1 min (RH 5 53.5%; 24°C). The accuracy of t1 and t2 (from
61 to 65 min, depending on the experiment) corresponded to the time interval between two photos. Scale bar, 10 mm.
did not control transpiration, the resistance to water
movement from the cell interior to the air being mainly
located in the air (Fig. 8A). The same result was
obtained when the experiments were performed with
radish (Raphanus sativus) hypocotyls, suggesting that it
was valid for all kinds of evaporating cell walls.
At first sight, our findings that cells lacking either
one of two AQPs showed an increased rate of wilting
may then seem counterintuitive and led us to analyze
our experimental conditions more closely.
Environmental Conditions of a Transpiring Leaf
In the nonvascularized, one-cell-thick leaves of P.
patens, each cell can exchange water with the external
medium. If water potential values in the external
medium, Ce, are the same on each side of the leaf
(Fig. 8B), the water potential within the cell, Ci, will
tend toward this value, corresponding to an equilibrium state without transpiration. There are two distinct cases: (1) the leaf is in contact with saturated air or
dilute solutions (condensation or rain) are in contact
with the leaf; Ce is close to 0 and the cells remain fully
turgid and sustain normal growth (Salisbury and Ross,
1992). (2) The leaf is transpiring in nonsaturated air
(Ce , 0); there is a loss of cellular water that can
produce wilting. In both cases the cell water status is
determined by the availability of water outside, not
by the permeability of the cell membranes. This may
explain the similar behavior observed between our
mutants lacking AQPs and wild type; all the plants
were turgid in closed petri dishes (no stress condition,
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Aquaporins Play Physiological Role in Physcomitrella patens
Figure 8. Schematic drawing of transmembrane and transcellular
transpiration flow (side view). A, Transpiration from a cell surface.
The magnitude of the transmembrane flow (black arrow) is mainly
determined by the resistance in the boundary layer (left drawing).
Opening AQPs adds a pathway to water across the membrane, but does
not modify the magnitude of the transpiration flow (right drawing). mb,
Membrane; cw, cell wall; bl, boundary layer; dotted arrow, flow
through the lipid bilayer; white arrow, flow through AQPs. B, When
both sides of a cell are in contact with a water potential Ce in the
external medium, the cell water potential Ci reaches an equilibrium
value Ci 5 Ce. If Ce ; 0 (left) the cell remains turgid; if Ce is too
negative (right) the cell becomes wilted. C, Transcellular transpiration
flow from a cell with one side facing liquid water (Ce ; 0), the other
side facing the air. When the transpiration flow (black arrow) is not too
high (medium stress), AQPs can keep Ci close to zero by reducing the
resistance of the membrane (left). For the same transpiration flow, if
AQPs are removed from the membrane (right), the increase in resistance can produce a more dangerous drop in Ci.
Ce ; 0) or wilted on dry filter paper (strong water
stress conditions, too negative Ce).
In the experiments on wet filters, our moderate
stress conditions, we assume that parts of the leaves
were either in contact with liquid water (Ce 5 0) or
with nonsaturated air (Ce # 0). If a cell is in contact
with liquid water on one side and with nonsaturated
air on the other side a transcellular water flow is
established (Fig. 8C). For the cell membrane in contact
with liquid water on both sides, the resistance rm to
water movement between cell interior and the liquid
water outside is mainly due to the cell membranes (the
resistance of the cell wall can be ignored because it is
usually far smaller than that of the cell membranes;
Nobel, 1999). This resistance is reduced if AQPs are
present in the membranes. For the cell membrane in
contact with the air, as already explained, the resistance between the cell interior and the air is mainly
due to rbl, the resistance of the air (the boundary layer)
near this surface; rbl does not depend on AQPs and,
according to Tazawa and Okazaki (1997), rbl rm.
The different magnitude of rbl and rm has two
consequences: (1) the total resistance to the transcellular flow, rbl 1 2*rm, is nearly equal to rbl. It is not
modified by AQPs and cells of approximately the
same size, regardless of AQPs, should evaporate at the
same rate; (2) for the same evaporation rate, the water
potential inside the mutant and wild-type cells may be
different: the larger the rm the larger the drop in cell
water potential (Fig. 8). As a consequence, under
moderate water stress, the wild type could maintain
Ci close to zero, but the suppression of an AQP in
pip2;1 and pip2;2 mutants could produce a dangerous
drop in water potential.
We conclude from our experiments that PIP2;1 and
PIP2;2 code for functional AQPs. These transcripts
were specifically expressed in the wild-type gametophores. The mutants lacking these genes wilted more
easily, which means that their metabolism was suspended earlier than wild type under the same moderate stress conditions. The physiological advantage of
a delay in wilting is obvious.
One interesting discussion point is that what we
refer to as medium stress conditions may represent the
situation of cells in the stomatal chamber of higher
plant leaves, which are partly in contact with the
intercellular air space. Therefore, even if AQPs are not
required on the cell surface in contact with the air,
AQPs could delay cellular water loss by facilitating
water exchanges through the part of the cell surface in
contact with the liquid phase.
The protonema, which normally grows in liquid
medium, did not express PIP2;1 and PIP2;2. This is not
surprising if the two AQPs carry out functions related
to transpiration. Gametophores may have adapted
various strategies to delay wilting, and increased cell
membrane permeability to water could be one such
strategy.
According to Proctor and Tuba (2002), poikilohydry
is not the primitive starting point leading to homoio-
Plant Physiol. Vol. 146, 2008
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Liénard et al.
hydry, but an alternative strategy to cope with irregular water supply. When water is not easily available,
rather than wasting energy maintaining a low water
potential in their cells, poikilohydry plants suspend
their metabolism during these periods of time. Our
study suggests that more than 400 million years before
the evolution of vascular plants, P. patens was already
using AQPs to shorten these periods.
MATERIALS AND METHODS
Material
Plants of the moss Physcomitrella patens wild-type strain (Gransden) were
provided by Martine Gonneau (Institut National de la Recherche Agronomique). Plants were grown axenically in petri dishes (90- or 45-mm diameter)
containing a liquid culture medium called A#BCD-TES-NH41 (Wang et al.,
1980) solidified with 0.8% (w/v) agar. The petri dishes were wrapped with
parafilm, so that plants developed in a water-saturated atmosphere. They
were kept in a growth chamber at 23 to 25°C with a 16-h/8-h light/dark cycle.
Light (60–75 mmol m22 s21) was provided by TLD 33 and chromasoleil TLD 83
(Philips) fluorescent tubes. The plants were subcultured every 7 d.
Standard molecular techniques were used (Sambrook et al., 1989). Restriction enzymes, Taq polymerase, pGEM-T vector, and T4 DNA ligase were
obtained from Promega. Primers were custom-ordered from Proligos. PCR
was performed in a thermocycler (Eppendorf). DNA sequencing was performed with an automated LI-COR 4000 sequencer.
Bacteria
The Escherichia coli strain DH5a used for plasmid DNA propagation was
grown in 10% tryptone (DIFCO), 5% yeast extract (DIFCO), and 170 mmol L21
NaCl.
Cloning of Three Putative AQP Coding Sequences
The 35 coding sequences of Arabidopsis (Arabidopsis thaliana) AQPs
(Johanson et al., 2001) were used to screen the ESTs from the PHYSCObase
(http://moss.nibb.ac.jp) database. One of these ESTs, referred to as 18342754
in GenBank, showed a strong similarity (E-value 5 10215) with the 5# end
of PIP2 AQPs. To amplify this partial sequence, two specific primers
were designed: EST1For (5#-ggCTgTggAgCggCACTAgA-3#) and EST1Rev
(5#-TggCAgAgAAgACggTgTAC-3#). PCR was carried out using total plant
cDNA as the template. Total RNAs were isolated using the SV 96 total RNA
isolation system kit (Promega) and reverse-transcribed with moloney murine
leukemia virus reverse transcriptase RNase H Minus (Promega) according to
the manufacturer’s instructions. Two different sequences were amplified. To
obtain the full-length coding sequence of each gene, 3#-RACE-PCR experiments were performed with the BD SMART mRNA amplification kit (BD
Biosciences CLONTECH), according to the manufacturer’s instructions. The
primers used were: EST1Race (5#-ggCATATCAACCCAgCAgTCACgTTC-3#)
and EST1BisRace (5#-ggCTTgCTgTTggCACgCAAAATCTC-3#). Once the 3#
cDNA ends were determined by RACE, gene-specific primers were designed
to amplify the entire cDNA from the start-to-stop codons and then to amplify
genomic sequences. PIP2;1 forward (5#-AgCAAACCATggCAAAAgAC-3#)
and reverse (5#-gATggAggAACTCgTgCTTA-3#) primers and PIP2;2 forward
(5#-AgCAAACCATggCAAAAgAC-3#) and reverse (5#-TgTCACgTTTAgATgTgACT-3#) primers were used.
The reversed sequence of a second EST (18334457 in GenBank) showed a
strong similarity (E-value 5 5 3 10230) with the 3# end of PIP2 AQPs. We
obtained the full-length cDNA by following the same procedure as described
above for EST 18342754. Two specific primers, EST2For (5#-CTggTgTACACCgTTTTCTC-3#) and EST2Rev (5#-TTgAACAggAgCAAATgCCC-3#),
were designed to amplify a partial sequence and an EST2Race primer
(5#-TgCCAgTTCCggTAATgggAATggTAg-3#) was used to determine the
5# cDNA end. Two gene-specific primers, PIP2;3 forward (5#-ggTTTTgCgAggAAgAAgTT-3#) and reverse (5#-ggAATTTgTgAgggggCAAg-3#), allowed
amplification of the entire cDNA.
The ClustalW program (Thompson et al., 1994) at http://www.infobiogen.
fr/services/analyseq/cgi-bin/clustalw_in.pl. was used for sequence alignments.
Knockout Constructs for PIP2;1, PIP2;2, and PIP2;3
The pNeo plasmid was constructed as follows. The EcoRI fragment from
pHP23b (Paszkowski et al., 1988), containing the neomycin phosphotransferase gene driven by the 35S cauliflower mosaic virus promoter, was removed
and ligated into the EcoRI site of the pMCS5 vector (MoBiTec), generating
pNeo with the neomycin phosphotransferase gene flanked by a multiple
cloning site.
Knockout plasmids were constructed (Fig. 5) by enzymatic digestion of the
three genes, and ligation into the pNeo plasmid. Each gene was cut with two
different sets of enzymes to produce two fragments, which were then cloned
into pNeo. PIP2;1 was digested with HpaII/StuI and BstZ17I/MluI to give two
fragments: one of 617 bp and one of 684 bp (corresponding to 624 bp of the
genomic sequence). These fragments were ligated in pNeo linearized by
BstBI/NruI and SpeI/MluI for the first and second fragments, respectively.
PIP2;2 was digested with BmgBI/AclI and NheI/BfrBI, to give two fragments:
one of 760 bp and one of 638 bp (corresponding to 577 bp of the genomic
sequence). These fragments were ligated in pNeo linearized by SmaI/BstBI
and SpeI/BfrBI for the first and second fragments, respectively. For PIP2;3 two
fragments (679 and 568 bp) were produced by digestion with ClaI/PmlI and
SpeI/XbaI. These fragments were ligated in pNeo linearized by BstBI/NruI
and SpeI/NsiI for the first and second fragments, respectively. These plasmids
were amplified and linearized by ScaI for transformation. Linear DNA was
purified by the Wizard SV Gel and PCR Clean-Up System from Promega, and
then resuspended in sterile water at a concentration of 0.5 mg/mL.
Transformation Procedure
Protoplasts were isolated from a 7-d-old protonematal culture by incubation for 30 min in 1% driselase (D8037; Sigma) and dissolved in 0.48 M
mannitol as previously described (Schaefer and Zryd, 1997). Transformation
experiments were performed with 300 mL of protoplast suspension added to
20 mL of linearized DNA (prepared as described above), using the standard
protocol (Schaefer and Zryd, 1997). After 7 d of incubation, regenerating
colonies were transferred to NH4 medium supplemented with 50 mg/mL of
paromomycin (P8692; Sigma). Stable antibiotic resistant clones were selected
by a second round of growth of fragmented plants on a Pp-NH4 medium
containing the antibiotic.
PCR Screening of Mutant Plants
For PCR analysis, genomic DNA was extracted using a genomic DNA
quick preparation for PCR method. Fresh tissue (50 mg) was ground in 300 mL
of extraction buffer (200 mM Tris-HCl, pH 7.5, 250 mM NaCl, 25 mM EDTA, and
0.5% SDS) and centrifuged at 12,000 rpm for 1 min. Isopropanol (200 mL) was
added to the supernatant, left for 2 min at room temperature, and centrifuged
at 12,000 rpm for 5 min. The pellet was resuspended in 100 mL of Tris EDTA
buffer; 1 mL was used for the PCR. Three primers for the PIP2;1 genomic locus
(Up21-2, 5#-TTTTCgTgTTggTgTACTgC-3#, PIP2;1 reverse, and Down21-8,
5#-CACTgCAAgTCACCAgAACT-3#), three primers for the PIP2;2 genomic locus
(Up22-2, 5#-AgCAgTCATCgCTgAgTTTg-3#, PIP2;2 reverse, and Down22-8,
5#-gCATAgTCACCAAggCTggT-3#), and two primers for the PIP2;3 genomic
locus (PIP2;3 forward and PIP2;3 reverse) were used in combination with
two nptII gene-specific primers (neo4, 5#-ATgAACTgTTCgCCAgTCTT-3# and
neo6, 5#-CCTAAAACCAAAATCCAgTg-3#).
Protoplasts Used for Pos Measurements
Plants from 2- to 3-week-old subcultures, grown on solid medium, were
used to prepare protoplasts for the permeability measurements. Protoplasts
from the protonema or gametophore leaves were used in the experiments. A
few strands of protonema were collected under a zoom microscope. They
were almost completely digested after 90 min at 28°C in the following
solution: driselase 1% (Sigma), mannitol 500 mmol/kg, macerozyme 0.1%
(Yakult Honsha), polyvinylpyrrolidone 0.5%, cellulase RS 0.5% (Yakult Honsha),
5 mM MES buffered with Tris to pH 5.5. When gametophores (5–10) were taken
from the same cultures, no protoplast was released after 90 min in this
solution. The digestion had to be extended for 20 h at room temperature under
sterile conditions to release enough protoplasts for our experiments.
Pos Measurements
Isolated protoplasts were resuspended in a storing solution (mannitol, 500
mmol/kg; Ca(No3)2, 1 mM; bovine serum albumin, 0.05% w/v; MES, 5 mM,
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Aquaporins Play Physiological Role in Physcomitrella patens
buffered with Tris to pH 5.5), and Pos was measured at room temperature
according to a technique previously described (Ramahaleo et al., 1999); the
value of Pos was deduced from the initial rate of swelling of the protoplast,
when transferred from the stock solution to a solution with lower osmoticum
(mannitol, 100–300 mmol/kg; Ca(No3)2, 1 mM; bovine serum albumin, 0.05%
w/v; MES, 5 mM, buffered with Tris to pH 5.5).
When the protonema from the wild type was digested for 90 min, the
measured protoplasts had a median value of Pos 5 2.7 mm s21 (Fig. 6A).
To detect the possible effect of a long incubation time on Pos, some protoplasts (10 from two different preparations) of the wild-type protonema were
kept for 20 h in the digestion solution, before measurements. Under these
conditions, the median value was 34.3 mm s21. The long incubation time
required to obtain protoplasts from the gametophores could therefore have
led to an overestimation of their Pos (median value is 159 mm s21). However,
the ANOVA test on ranks indicated that the large increase in Pos from the
protonema to the gametophore could not only be explained by longer
digestion.
Water Stress
Isolated mutant and wild-type gametophores were grown in 100% RH and
then subjected to water stress by exposure to lower RH from ambient air in the
laboratory. The parameters of the laboratory environment (RH, 49% to 60%;
temperature, 24°C to 28°C) were not regulated but fluctuations were #1%
RH and #1°C within an experiment lasting 3 h or less. A surface mimicking
wet ground evaporating in air was made by depositing a Whatmann filter
paper disc (diameter 40 mm) on an open petri dish filled with a 1% agar
solution (w/v) in water. When the dish was placed on the stage of an M3Z
stereomicroscope (Leica), the paper remained wet for several hours under
laboratory conditions. A dry filter paper disc (diameter 40 mm) was used to
mimic dry ground. Gametophores of approximately the same size (see Fig. 7,
A to C) were cut from 8-week-old wild-type and mutant plants. Immediately
after depositing the gametophores on the filters, photos were taken every
1 to 5 min for 2 to 3 h, using a camera (EOS 300D; Canon) with an adapter
for the stereomicroscope. Plants were exposed to a 1,300-lux cold light (KL
1500 LCD; Leica).
Software
The distribution of Pos values for all the groups of wild-type and mutant
protoplasts did not always follow a normal distribution (the SD was about the
same size as the mean) so we used nonparametric tests (Glantz, 1997) to
compare the four groups: wild type, pip2;1, pip2;2, and pip2;3. Each Pos value
was given a rank: the smallest was ranked 1. The next biggest ranked 2 and so
on, up to the largest value. All the values were ranked without regard for the
group from which they were derived. If wild type and mutant Pos values were
the same, the average rank for each group should be similar to the average
rank computed without regard for the grouping.
We used the nonparametric Dunn’s test and one-way ANOVA on ranks
test from the program SigmaStat (Systat Software GmbH) to identify significant differences between average ranks, i.e. between the groups. The graphs
were drawn with SigmaPlot (Systat Software GmbH).
Sequence data from this article have been deposited with the EMBL/
GenBank database/ libraries under accession numbers AY494191 (PIP2;1),
AY494192 (PIP2;2), and DQ018113 (PIP2;3).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Video S1. Wilting of a gametophore deposited on a wet
filter (associated with Figure 7A; 1 s on the video corresponds to 5 min
in real time).
Supplemental Video S2. Wilting of a gametophore deposited on a wet
filter (associated with Figure 7B; 1 s on the video corresponds to 3 min
in real time).
Supplemental Video S3. Wilting of a gametophore deposited on a dry
filter (associated with Figure 7C; 1 s on the video corresponds to 1 min
in real time).
ACKNOWLEDGMENTS
We thank Martine Gonneau for introducing us to P. patens. We thank
Professor Hervé Dupont, Dr. Loı̈c Faye, and Professor Susannah Gal for their
critical comments on the manuscript.
Received October 19, 2007; accepted December 20, 2007; published January 9,
2008.
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